This proposal has two main aspects, focused on the function and evolution of reproductive genes. First, we will use a novel proteomics approach to identify the seminal fluid components that are transferred from males to females during mating in a variety of Drosophila species. This approach will utilize the genomic data from the twelve Drosophila genomes sequencing project, allowing for the investigation of the evolutionary dynamics of these genes. We anticipate finding evidence for adaptive evolution. Additionally, we expect to uncover dynamic gene gain/loss. From these analyses, we will gain insights into the evolution of a unique class of reproductive proteins. A common finding in genome sequencing projects is the rapid evolution of reproductive genes. However, these genes are usually predicted to be involved in reproduction based only upon tissue expression or indirect evidence. By combining our proteomic data with genomic resources, we will have a unique set of reproductive genes to investigate this general observation. The second aspect of our proposal is to investigate the functional differentiation of these genes, using both sequence data and quantitative mass spectrometry. Drosophila seminal fluid is implicated in many aspects of sperm competition. Additionally, heritable genetic variation exists within populations for these traits. However, few associations between seminal fluid genes and sperm competition phenotype have been reported. We will utilize a clonal method to generate hemiclonal males from an outbred population and screen these flies for the extreme tails of sperm competitiveness. These lines will be analyzed for both seminal fluid gene sequence variation and quantity of each seminal fluid protein transferred. The latter is a novel approach and directly measures the relevant biological trait (quantity of protein transferred to females), rather than the typical approach of measuring transcript abundance in male flies. Relevance to public health: Genome sequencing projects repeatedly find reproductive genes to be the most divergent genes from a wide variety of organisms, including humans. A complete molecular understanding of reproduction and fertilization requires investigation into why these genes are evolving so rapidly. While the proposal focuses on Drosophila, similar classes of genes and evolutionary dynamics have been observed in primate seminal fluid. Therefore, the results here may provide insights into primate reproductive biology.